Publications

3996

Abstract The reference strain of 'Ca. Phytoplasma trifolii' is the causative agent of clover proliferation (CP) disease of alsike clover (Trifolium hybridum). The CP disease was first reported in Canada in the early 1960s when the aetiological agent was mistakenly presumed to be a yellows-type virus (Chiykowski, 1965). Subsequent investigations revealed that the disease was associated with infection by a mycoplasma-like organism (Chen and Hiruki, 1975; Hiruki and Chen, 1984), now termed phytoplasma, strain CPR (Hiruki and Wang, 2004). Later, phytoplasmas of the same lineage (subgroup 16SrVI-A) were found in the USA, Mexico, and many countries in Europe and Asia, causing diseases in diverse leguminous and vegetable crops, responsible for significant yield losses and quality reductions. Phytoplasmas of the same lineage also caused disease in elm trees in the USA. Phytoplasmas of closely-related lineages (various subgroups of group 16SrVI) also have wide distributions around the world.

Understanding temporal biological phenomena is a challenging task that can be approached using network analysis. Here, we explored whether network reconstruction can be used to better understand the temporal dynamics of bois noir, which is associated with ‘Candidatus Phytoplasma solani’, and is one of the most widespread phytoplasma diseases of grapevine in Europe. We proposed a methodology that explores the temporal network dynamics at the community level, i.e., densely connected subnetworks. The methodology offers both insights into the functional dynamics via enrichment analysis at the community level, and analyses of the community dissipation, as a measure that accounts for community degradation. We validated this methodology with cases on experimental temporal expression data of uninfected grapevines and grapevines infected with ‘Ca. P. solani’. These data confirm some known gene communities involved in this infection. They also reveal several new gene communities and their potential regulatory networks that have not been linked to ‘Ca. P. solani’ to date. To confirm the capabilities of the proposed method, selected predictions were empirically evaluated.

Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with ‘Candidatus Phytoplasma solani’, but molecular interactions between the causal pathogen and its host plant are not well understood. In this work, we combined the analysis of high-throughput RNA-Seq and sRNA-Seq data with interaction network analysis for finding new cross-talks among pathways involved in infection of grapevine cv. Zweigelt with ‘Ca. P. solani’ in early and late growing seasons. While the early growing season was very dynamic at the transcriptional level in asymptomatic grapevines, the regulation at the level of small RNAs was more pronounced later in the season when symptoms developed in infected grapevines. Most differentially expressed small RNAs were associated with biotic stress. Our study also exposes the less-studied role of hormones in disease development and shows that hormonal balance was already perturbed before symptoms development in infected grapevines. Analysis at the level of communities of genes and mRNA-microRNA interaction networks revealed several new genes (e.g., expansins and cryptdin) that have not been associated with phytoplasma pathogenicity previously. These novel actors may present a new reference framework for research and diagnostics of phytoplasma diseases of grapevine.

Background: ‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus greening disease. The disease primarily involves an asymptomatic, often latent infection of CLas. However, there is no effective technique to distinguish latent-infected trees from healthy ones. This study describes the development of a new detection method for latent CLas infection using cuttings. Methods: Root tissues regenerated from cuttings using symptomatic and asymptomatic citrus trees were prepared for real-time a polymerase chain reaction (PCR) test which was used to investigate latent CLas. When some of the regenerated roots were negative for CLas in the first real-time PCR assay, a subsequent cultivation in soils was performed using the CLas-negative cuttings. CLas development during cultivation was evaluated by a second real-time PCR assay using soil-grown roots from seedlings. Results: Previously, CLas had not been detected from leaves of the latent-infected trees in our greenhouse by real-time PCR. In this study, however, CLas was detected at a moderate frequency from the root tissues of cuttings derived from the latent-infected trees, by the same PCR test. For cuttings with regenerated roots that tested negative for CLas by real-time PCR, CLas was frequently detected from roots grown in nursery soil with autoclaving, after cultivation for a month or more. Conclusions: Latent infection with CLas was detectable by real-time PCR using root tissues regenerated by cuttings and roots grown in nursery soil with autoclaving. These results suggest that the new method of investigation would provide great opportunities for early detection of CLas in asymptomatic citrus trees from field surveys, and would accelerate the eradication practice of citrus greening.

AbstractIn the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, non-phosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme’s affinity for UDP-glucose, which improves its catalytic efficiency. Homology modelling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling, but also describe an efficient enzymatic tool for future synthesis of bioactive molecules.ImportanceThe bilayer formed by membrane lipids serves as the containment unit for living microbial cells. In the marine environment, it has been firmly established that phytoplankton and heterotrophic bacteria can substitute phospholipids with non-phosphorus sugar-containing glycoglycerolipids in response to phosphorus limitation. However, little is known about how these glycoglycerolipids are synthesized. Here, we determined the biochemical characteristics of a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211. GTcp and its homologs form a group in the GT4 glycosyltransferase family, and can synthesize neutral glycolipids (MGlc-DAG and MGal-DAG) and an acidic glycolipid (MGlcA-DAG). We also uncover the key residues for DAG-binding through molecular docking, site-direct mutagenesis and subsequent enzyme activity assays. Our data provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling.

SummaryMicrobes are phylogenetically and metabolically diverse. Yet capturing this diversity, assigning functions to host organisms and exploring the biosynthetic potential in natural environments remains challenging. We reconstructed >25,000 draft genomes, including from >2,500 uncharacterized species, from globally-distributed ocean microbial communities, and combined them with ∼10,000 genomes from cultivated and single cells. Mining this resource revealed ∼40,000 putative biosynthetic gene clusters (BGCs), many from unknown phylogenetic groups. Among these, we discovered Candidatus Eudoremicrobiaceae as one of the most biosynthetically diverse microbes detected to date. Discrete transcriptional states structuring natural populations were associated with a potentially niche-partitioning role for BGC products. Together with the characterization of the first Eudoremicrobiaceae natural product, this study demonstrates how microbiomics enables prospecting for candidate bioactive compounds in underexplored microbes and environments.

AbstractCandidatus Izemoplasma, an intermediate in the reductive evolution from Firmicutes to Mollicutes, was proposed to represent a novel class of free-living wall-less bacteria within the phylum Tenericutes. Unfortunately, the paucity of pure cultures has limited further insights into their physiological and metabolic features as well as ecological roles. Here, we report the first successful isolation of an Izemoplasma representative from the deep-sea methane seep, strain zrk13, using a DNA degradation-driven method given Izemoplasma’s prominent DNA-degradation potentials. We further present a detailed description of the physiological, genomic and metabolic traits of the novel strain, which allows for the first time the reconstruction of the metabolic potential and lifestyle of a member of the tentatively defined Candidatus Izemoplasma. On the basis of the description of strain zrk13, the novel species and genus Xianfuyuplasma coldseepsis is proposed. Using a combined biochemical and transcriptomic method, we further show the supplement of organic matter, thiosulfate or bacterial genomic DNA could evidently promote the growth of strain zrk13. In particular, strain zrk13 could degrade and utilize the extracellular DNA for growth in both laboraterial and deep-sea conditions. Moreover, the predicted genes determining DNA-degradation broadly distribute in the genomes of other Izemoplasma members. Given that extracellular DNA is a particularly crucial phosphorus as well as nitrogen and carbon source for microorganisms in the seafloor, Izemoplasma bacteria are thought to be important contributors to the biogeochemical cycling in the deep ocean.

AbstractMitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis1,2. Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution3. As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation4. Here we describe ‘Candidatus Azoamicus ciliaticola’, which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. ‘Candidatus A. ciliaticola’ contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron–sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. ‘Candidatus A. ciliaticola’ and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.